Generating cardiovascular cells from pluripotent stem cells holds great promise for cardiovascular research and therapy. However, cardiogenesis is regulated by numerous developmental pathways. Moreover, differentiation of pluripotent stem cell into cardiac cells is inefficient and results in heterogeneous cultures, limiting the usefulness of this approach. Pluripotent stem cells, such as human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells, collectively human pluripotent (hPS) cells can perpetually proliferate and differentiate into derivatives of all three embryonic germ layers (Thomson et al., Science 282:1145 (1998); Odorico et al., Stem Cells 19:193 (2001); Yu et al., Science 318(5858):1917 (2007)). Pluripotent stem cell cultures can differentiate spontaneously, yielding a seemingly random variety of cells (Watt and Hogan, Science 287:1427 (2000)). The earliest pluripotent stem cell differentiation methods allowed stem cell aggregates to spontaneously differentiate and form embryoid bodies (EBs) which contain precursors of the three primary germ layers, including, in some cases, cardiomyocytes. Such methods are inefficient, however, as only few percent of the developing cells become cardiomyocytes.
More recent methods direct differentiation of ES and iPS cells (pluripotent cells generated by reprogramming somatic cells or differentiated progenitor cells to pluripotency) into cardiomyocytes without EB formation by sequentially applying various combinations of soluble, exogenous growth factors and small molecules to mimic cardiac development. Soluble factors important for embryonic cardiac development include Activin A, BMP4, nodal, Wnt agonists and antagonists, bFGF and other molecules (Conlon et al., Development 120(7):1919 (1994); Lough et al., Dev. Biol. 178(1):198 (1996); Mima et al., PNAS 92(2):467 (1995); Zaffran and Frasch, Circ. Res. 91 (6), 457 (2002)). The addition of FGF2, Activin A, BMP4, DKK1 and VEGF, can enhance cardiomyocyte differentiation in embryoid bodies (EBs) (Yang et al., Nature 453:524-528 (2008)). However, this protocol is labor-intensive and not applicable to all pluripotent cell lines since it requires monitoring of KDR/c-kit (Yang et al., Nature 453:524-528 (2008)) or Flk1/PDGFRα (Kattman et al., Cell Stem Cel/8:228-240 (2011)) expression and optimization of growth factor concentrations for efficient cardiac development in various hPSC lines. Protocols for cardiomyocyte progenitor and cardiomyocyte differentiation that do not require cell line-specific optimization are desirable. Identification of defined factors that promote cardiomyocyte progenitor and cardiomyocyte differentiation has enabled development of monolayer-based directed differentiation protocols, such as, sequential treatment of Activin A and BMP4, which has been reported to generate greater than 30% cardiomyocytes in the H7 hESC line (Laflamme et al., Nat. Biotechnol. 25:1015-1024 (2007)). However, the efficiency of the Activin A and BMP4 directed differentiation protocol can be highly variable between cell lines and experimental repeats (Paige et al., PLoS One 5: e11134 (2010)).
Apart from their somatic cell origin, iPS cells share many characteristics of embryonic stem cells, such as the ability to grow perpetually and to differentiate into cells of all three germ layers. Like ES cells, iPS cells express pluripotency markers, such as OCT-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Nanog. iPS cells have been generated using retroviral vectors that randomly insert exogenous DNA into the target cell genome. Vector- and transgene-free iPS cells have been generated by using non-integrating vectors. Using non-integrating vectors avoids the risk of aberrant cellular gene expression and neoplastic growth (Okita et al. Nature 448:313 (2007)). Loss of the reprogramming vector also avoids perpetual expression of transgenes that can induce programmed cell death (apoptosis) (Askew et al., Oncogene 6:1915 (1991), Evan et al., Cell 69:119 (1992)) and interfere with subsequent differentiation of iPS cells.
More recently, methods were devised for reprogramming somatic cells using oriP/Epstein-Barr nuclear antigen-1 (EBNA1)-based episomal vectors that do not integrate into the genome and are lost from the cells after reprogramming (Yu et al., Science 324(5928):797 (2009)). iPS cells generated by this method are vector- and transgene-free and, as such, are well suited for clinical application. However, vector-free iPS cells have not yet been demonstrated to differentiate into clinically-applicable cardiomyocyte progenitors and cardiomyocytes with high efficiency (e.g., >90%) under fully defined conditions (both medium and substrate are defined).
Wnt proteins control morphogenesis, and are involved in development, stem cell differentiation control, and cell malignant transformation. The Wnt signaling pathway is a key regulator of cardiogenesis in vivo and in vitro. In chick and frog embryos, canonical Wnt signaling represses early cardiac specification (Marvin et al., Genes Dev. 15, 316-327 (2001); Schneider and Mercola, Genes Dev. 15, 304-315 (2001); Tzahor and Lassar, Genes Dev 15, 255-260 (2001)) and has a biphasic effect in zebrafish, mouse embryos, and mouse embryonic stem cells (Naito et al., Proc. Natl Acad. Sci. U.S.A. 103, 19812-19817 (2006); Ueno et al., Proc. Natl. Acad. Sci. U.S.A. 104, 9685-9690 (2007), with early Wnt signaling enhancing cardiogenesis and later signaling repressing heart development. Endogenous Wnt signaling is also required after treatment to differentiate hES cells to cardiomyocytes with Activin A and BMP4 (Paige et al., PLoS One 5: e11134 (2010)). However, it remains unknown whether differentiation of human pluripotent stem cells to cardiomyoctes conserves such stage-specific Wnt signaling roles.
The canonical Wnt pathway describes a series of events that occur when Wnt ligands bind to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins, resulting in a change in the amount of β-catenin that reaches the nucleus. Modulation of Gsk3 and Wnt pathway signaling triggers expression of a variety of developmental cues (e.g. Nodal (Kattman et al., Cell Stem Cell, 8:228-240 (2011)), BMP2/4 (Kattman et al., 2011; Laflamme et al., Nat. Biotech 25:1015-1024 (2007)), Noggin (Ma et al., Cell Res. 21:579-587 (2011)), WNT3a (Tran et al., Stem Cells, 27:1869-1878 (2009)) and WNT8a (Paige et al., PLoS One 5:e11134 (2010)) and transcription factors involved in cardiomyocyte differentiation (e.g. T (Asashima et al., Faseb J. 23:114-122(2009)) and MIXL1 (Davis et al., Blood 111:1876-1884 (2008)), ISL1 (Bu et al., Nature 460:113-117 (2009) and NKX2-5 (Lints et al., Development 119:969 (1993)), TBX5 (Bruneau et al., Dev Biol 211:100-108 (1999)), GATA4 (Kuo et al., Gene Dev 11:1048-1060 (1997a); Kuo et al., Circulation 96:1686-1686 (1997), and MEF2C (Edmondson et al., Development 120:1251-1263 (1994)). There is in the art a need for a cardiac differentiation protocol that uses completely defined, growth factor-free culture conditions to produce cardiomyocyte progenitors and cardiomyocytes from hPS cells.